Method for preparing heparin from mast cell cultures

ABSTRACT

The invention concerns the production of heparin from mast cell cultures, in particular pig mast cells.

The present invention relates to the preparation of heparin from cellcultures.

Heparin belongs to the glycosaminoglycan (GAG) family, which includesthe linear polysaccharides containing a repeat of a disaccharidesequence made up of an amino sugar (D-glucosamine or galactosamine) anda uronic acid (D-glucuronic or iduronic).

In the case of heparin, which belongs, with heparan sulfate, to theglucosaminoglycan subfamily, the amino sugar is D-glucosamine. Theuronic acid is either glucuronic acid (Glc) or iduronic acid (Ido). Theglucosamine can be N-acetylated, N-sulfated or O-sulfated.

Conventionally, the term “heparin” refers to highly sulfatedpolysaccharides in which more than 80% of the glucosamine residues areN-sulfated and the number of O-sulfates is greater than that of theN-sulfates. The sulfate/disaccharide ratio is generally greater than 2for heparin. However, the structure of heparin is in fact veryheterogeneous, and chains which can contain very different ratios exist.

Like all GAGs, heparin is synthesized in the form of a proteoglycan.This synthesis takes place preferentially in a subpopulation of mastcells, serous or connective tissue mast cells (CTMCs). These mast cellsare abundant in the skin and the respiratory submucosae. They have avery long lifespan (at least 6 months). Besides heparin, they containheparan sulfate and appreciable amounts of histamine (approximately 10pg/cell, according to the animal species).

The first step of heparin synthesis is the formation of the serglycineprotein core consisting of regularly alternating serine and glycineresidues. Elongation of the heparin chain takes place from atetrasaccharide, by successive additions of osamine and of uronic acids.

The proteoglycan thus formed undergoes many sequential transformations:N-deacetylation, N-sulfation, D-glucuronic acid epimerization, andO-sulfation.

However, this complete maturation only takes place on part of theproteoglycan, which generates a great structural variability of heparin,responsible for its heterogeneity.

The polysaccharide chains are then cleaved from the serglycine by anendoglucuronidase. These chains then have a molecular weight of between5 000 and 30 000 Da. They form complexes with alkaline proteases and arethus stored in the mast cell granules. Heparin is excreted only duringmast cell degranulation.

Heparin plays an important biological role, in particular in hemostasis,and is very widely used in therapeutics, in particular as ananticoagulant and an antithrombotic agent.

Currently, most of the heparin used is isolated from pig intestinalmucosa, from where it is extracted by proteolysis, followed bypurification on anion exchange resin (for a review on the variousmethods for preparing heparin, cf. DUCLOS; “L'Héparine: fabrication,structure, proprietes, analyse”; Ed. Masson, Paris, 1984).

Added to the inherent heterogeneity of heparin is the diversity of thebatches of animals from which it is obtained. A very substantialvariability results therefrom, reflected in particular in the level ofbiological activity. In addition, it is difficult to regularly have asufficient supply of raw material.

The use of cells derived from mammals for producing GAG or proteoglycanshas already been proposed.

Thus, application WO 99/26983 describes the obtaining of compounds ofthe heparin type, which may be proteoglycans (HEP-PG) orglycosaminoglycans (HEP-GAG), from rat mast cells. The compounds are notheparin. The cells thus isolated are not established lines. In addition,the applicant recommends coculturing the isolated cells withfibroblasts.

The article by Wang and Kovanen (Circulation Research, 84, 1, 74-83,1999) itself also describes the isolation of rat serous mast cells andthe production of proteoglycans from these cells. As in application WO99/26983, the cells used for the production of proteoglycans are notestablished lines, but simply cells which have been isolated and thenstimulated to produce proteoglycans.

Application WO 90/14418, cited in the search report, describes celllines obtained from mouse mastocytomas and their use for the productionof heparin. The origin of these cells is therefore tumoral, which mayraise health problems. An article by Montgomery et al. (Proc Natl AcadSci USA, 89, 23, 11327-11331, 1992) itself also describes the isolationof mouse mastocytomas.

The present invention proposes to overcome the drawbacks mentioned aboveand to avoid problems of supply in terms of quantity and of quality,using a conveniently available source of homogeneous raw material, withstable characteristics, facilitating the production of preparations ofheparin of constant quality.

The inventors have noted that it is possible to produce, from mast cellline cultures, a considerable amount of heparin having propertiescomparable to those of the heparin extracted from pig intestinal mucosa.The use of cell cultures as raw material also makes it possible tocontrol the conditions for synthesizing the heparin, and to thus obtaina product having reproducible characteristics.

A subject of the present invention is a method for producing heparin,characterized in that it comprises culturing mast cells of porcineorigin and recovering the heparin from the cultures obtained.

Preferably, said mast cell cultures are mast cell lines of porcineorigin.

The term “culture” here denotes, in general, a cell or a set of cellsgrown in vitro. A culture developed directly from a cell or tissuesample taken from an animal is referred to as a “primary culture”. Theterm “line” is employed when at least one passage, and generally severalconsecutive passages in subculture, have been successfully performed,and denotes any culture which is derived therefrom (SCHAEFFER, In VitroCellular and Developmental Biology, 26, 91-101, 1990).

Advantageously, said mast cells are derived from porcine mast cellcultures and in particular from porcine mast cell lines obtained asdescribed in Application FR 0113608, and also in the PCT applicationentitled “Cultures de mastocytes de porc et leurs utilisations” [pigmast cell cultures and their uses] in the name of INRA and of ENVA filedon the same day as the present application. Among these, preferred linesfor implementing the method in accordance with the invention are:

-   -   the mast cell line derived from pig fetal liver deposited by        INRA (147 rue de l'Université, 75007 Paris, France) with the        CNCM (Collection Nationale de Cultures de Microorganismes        [National Collection of Cultures of Microorganisms], Pasteur        Institute, 26, rue du Docteur Roux, 75724 PARIS CEDEX 15,        France) on Oct. 17, 2001, under the number I-2735;    -   the mast cell line derived from pig fetal liver and transfected        with the SV40 virus T antigen, deposited by INRA with the CNCM        on Oct. 17, 2001, under the number I-2736;    -   the mast cell line derived from pig fetal bone marrow and        transfected with the SV40 virus T antigen, deposited by INRA        with the CNCM on Oct. 17, 2001, under the number I-2734.

Preferably, these mast cells are serous mast cells.

These mast cells will preferably be cultured in a defined culture medium(MEMα/DMEM, RPMI, IMDM, etc.) supplemented with growth factors, used incombination or individually, such as SCF (Stem Cell Factor) at aconcentration of between 1 ng/ml and 1 μg/ml and, optionally, IL3(interleukin 3) at a concentration of between 0.1 ng/ml and 100 ng/ml,or PGE2 (prostaglandin E2) at a concentration of between 1 nM and 1 μM.

The media may also be supplemented with bovine serum, at a concentrationof between 0.5% and 20% (v/v).

The addition of bovine serum to the culture media can be replaced withthe use of a serum-free culture medium such as AIMV (INVITROGEN) so asto reduce the protein concentration of the medium and the risksassociated with the use of compounds of animal origin (KAMBE et al., J.Immunol. Methods, 240, 101-10, 200).

It is possible to obtain cells which do not depend on the addition ofserum and/or the use of growth factors by controlled mutation of thecell phenotype through the action of transformer and/or immortalizingagents (TSUJIMURA, Pathology International, 46, 933-8, 1996; PIAO andBERNSTEIN, Blood, 87(8), 3117-23, 1996).

The mast cells can be cultured using the techniques developed for themass culture of eukaryotic cells, as described, for example, byGRIFFITHS et al. (Animal Cell Biology, Eds. Spier and Griffiths,Academic Press, London, Vol. 3, 179-220, 1986). It is possible to usebioreactors with a volume greater than several m³, as described byPHILIPS et al. (Large Scale Mammalian Cell Culture, Eds. Feder andTolbert, Academic Press, Orlando, USA, 1985) or by MIZRAHI (ProcessBiochem, Aug. 9-12, 1983).

The culturing can also be carried out in suspension or on a microsupportaccording to the technique described by VAN MEZEL (Nature, 216, 64-65,1967).

It is also possible to use batch culturing systems, which are commonlyused for eukaryotic cell cultures due to the fact that they are muchsimpler to use on an industrial scale (VOGEL and TODARO, Fermentationand Biochemical Engineering Handbook, 2^(nd) edition, Noyes Publication,Westwood, N.J., USA, 1997). The cell densities obtained with thesesystems are generally between 10⁶ and 5×10⁶ cells/ml.

The productivity of the batch cultures can advantageously be increasedby removing some of the cells from the bioreactor (70% to 90%) for theGAG extraction and heparin isolation operations, and keeping theremaining cells within the same bioreactor in order to initiate a newculture. In this “repeated batch” culturing mode, it is also possible todistinguish the optimum parameters of the cell growth phase from thosewhich allow greater accumulation of GAGs and of heparin within thecells.

Continuous perfusion-fed culture systems, with or without cellretention, can also be used (VELEZ at al., J. Immunol. Methods, 102(2),275-278, 1987; CHAUBARD et al., Gen. Eng. News, 20, 18-48, 2000). In thecontext of the present invention, use may in particular be made ofperfusion-fed culture systems which allow cells to be retained withinthe reactor, and which result in a growth and a production greater thanthose which can be obtained in batch culture. The retention can beeffected by virtue of retention systems of the spin-filter, hollow fiberor solid matrix type (WANG et al., Cytotechnology, 9, 41-49, 1992; VELEZet al., J. Immunol. Methods, 102(2), 275-278, 1987). The cell densitiesobtained are generally between 10⁷ and 5×10⁷ cells/ml. Culturing inbioreactors allows, through the use of on-line measuring sensors, bettercontrol of the physicochemical parameters of the cell growth and also ofthe accumulation of GAGs and of heparin within the cells: pH, PO₂,Red/Ox, growth substrates such as vitamins, amino acids, carbon-basedsubstrates (for example glucose, fructose, galactose), metabolites suchas lactate or aqueous ammonia, etc.

The cells can be harvested and separated from the culture medium,generally by centrifugation or filtration, after from 3 to 30 days ofculturing, generally after from 3 to 10 days of culturing, under theseconditions.

Various centrifugation systems can be used; mention will, for example,be made of those described by VOGEL and TODARO (Fermentation andBiochemical Engineering Handbook, 2^(nd) Edition, Noyes Publication,Westwood, N.J., USA).

Alternatively, or in combination with centrifugation, the separation maybe carried out by tangential microfiltration using membranes theporosity of which is less than the average diameter of the cells (5 to20 μm) while at the same time allowing the other compounds insolution/suspension to pass through. The rate of tangential flow and thepressure applied to the membrane will be chosen so as to generate littleshear force (Reynolds number less than 5 000 sec⁻¹) in order to reduceclogging of the membranes and to preserve the integrity of the cellsduring the separating operation.

Various membranes can be used, for example spiral membranes (AMICON,MILLIPORE), flat membranes or hollow fibers (AMICON, MILLIPORE,SARTORIUS, PALL, GF).

It is also possible to choose membranes the porosity, the charge or thegrafting of which makes it possible to perform a separation and a firstpurification with respect to possible contaminants which may be presentin the culture medium, such as cell proteins, DNA, viruses, or othermacromolecules.

Use may be made of methods of production and of cell harvesting whichmake it possible to conserve the GAGs and the heparin in theintracellular content; however, the GAGs and the heparin can also beharvested from the culture medium after lysis or degranulation of thecells.

The degranulation may be caused by the binding of specific ligands tothe receptors present at the surface of the mast cells, for example thebinding of allergen-type agents (such as IgE Fc fragment or analogs ofthis fragment) to the mast cell IgE receptors. When the heparin has beenreleased from the intracellular content, by degranulation or lysis ofall or some of the mast cells, and is present in the culture medium atthe time of the separation step, the use of membranes with a smallerporosity may also be envisaged. In this case, the cell separation iscombined with a step consisting of ultrafiltration on one or moremembranes, the organization and the porosity of which make it possibleto concentrate the heparin and to separate it from the other speciespresent in the medium, as a function of the size and the molecularweight and, optionally, of the electrical charge, or of the biologicalproperties.

In the context of this embodiment, the cutoff threshold of the membranesis preferably between 1000 and 5 kDa. Use may be made of membranesystems similar to those used for microfiltration, for example spiralmembranes, flat membranes or hollow fibers. Use may advantageously bemade of membranes which make it possible to separate and purify theheparin due to their charge properties or their properties of graftingof ligands exhibiting affinity for heparin (for example antibodies,ATIII, lectin, peptides, nucleotides, etc.).

Other agents can also induce mast-cell degranulation. These agents canbe classified in several categories, such as cytotoxic agents, enzymes,polysaccharides, lectins, anaphylatoxins, basic compounds (compound48/80, substance P, etc.), calcium (A23187 ionophore, ionomycin, etc.)[D. Lagunoff and T. W. Martin, 1983, Agents that release histamine frommast cells. Ann. Rev. Pharmacol. Toxicol., 23:331-51]. A degranulatingagent can be used repeatedly on the same cells maintained in culture. Inthis method of production, the productivity is increased significantlyby the simplification of the method of harvesting from the supernatantand by the maintaining of the cells in culture.

In the particular case of A23187 ionophore, the mast-cell degranulationcan be induced, for example, by treatment of 2×10⁶ mast cells/ml withthe A23187 ionophore at concentrations between 1 and 100 μg/ml andaction times ranging from 1 minute to 4 hours.

The mast-cell lysis can be induced, for example, by osmotic shock usinghypotonic or hypertonic solutions, by thermal shock (freezing/thawing),by mechanical shock (for example sonication or pressure variation), bythe action of chemical agents (NaOH, THESIT™, NP40™, TWEEN 20™,BRIJ-58™, TRITON X™-100, etc.) or by enzyme lysis (papain, trypsin,etc.), or by a combination of two or more of these methods.

To extract and purify the heparin from the cell lysate, to separate thepolysaccharide chains from the serglycine core, and to separate theheparin chains from the other GAGs present in the extraction medium, usemay be made of methods similar to those used in the context of theextraction and purification of heparin from animal tissues, which areknown in themselves, and described in general works such as the manualby DUCLOS (mentioned above).

By way of nonlimiting examples, in order to separate the heparin fromthe nucleic acids and from the cell proteins, and to solubilize it, i.e.to break the bonds with the serglycine core:

-   -   the cell lysate can be subjected to one or more enzyme        digestions (pronase, trypsin, papain, etc.);    -   the heparin-protein bonds can be hydrolyzed in alkali medium, in        the presence of sulfates or chlorides;    -   it is also possible to carry out a treatment in acid medium (for        example with trichloroacetic acid under cold conditions) in        order to destroy the nucleic acids and the proteins originating        from the cells, to which is added the use of an ionic solution        which makes it possible to dissociate the GAG-protein        interactions;    -   it is also possible to carry out an extraction with guanidine,        after enzyme hydrolysis; to purify the solubilized heparin, it        is possible, for example, to precipitate it with potassium        acetate, with a quaternary ammonium, with acetone, etc.

These purification steps can advantageously have added to them or bereplaced with one or more chromatography steps, in particular anionexchange chromatography or affinity chromatography steps.

A subject of the present invention is also the heparin preparationswhich can be obtained from mast cell cultures using a method accordingto the invention.

The heparin preparations in accordance with the invention, which havebiological properties comparable to those of the heparin preparationsobtained in the prior art from animal tissues, can be used in all theusual applications for heparin.

The present invention will be understood more clearly from theadditional description which follows, which refers to examples ofpreparing heparin from mast cell cultures and of characterizing theheparin obtained.

EXAMPLE 1 Extraction of Heparin from Mast Cell Cultures

Culturing of Mast Cells

A pig fetal liver mast cell line and a line of pig fetal liver mastcells transfected with the SV40 virus T antigen (lines CNCM I-2735 andCNCM I-2736, respectively) were used.

The cells are seeded at a rate of 10⁵ to 5×10⁵ cells/ml, in completeMEMα medium in the presence of porcine IL3 (2 ng/ml) and of porcine SCF(80 ng/ml).

The cultures are prepared in a culture dish or in suspension in a1-liter spinner flask. The cell growth is monitored daily for 4 to 12days. The heparin production is monitored in parallel, by analyzing theglycosaminoglycans produced in culture. The results are given in FIGS. 1to 5.

FIGS. 1, 2 and 3 illustrate the growth of liver mast cells in staticculture in dishes (FIG. 1; initial seeding: ♦: 1×10⁵ cells; ▪: 2×10⁵cells) and in suspension in flasks (FIG. 2), and the growth oftransfected liver mast cells in suspension in flasks (FIG. 3).

In these experiments, the cultures in suspension in flasks exhibit amaximum cell density ranging from approximately 8×10⁵ (for thenontransfected cells) to approximately 1.5×10⁶ cells/ml (for thetransfected cells). The doubling time, calculated during the exponentialgrowth phase, is between 24 and 48 hours.

Glycosaminoglycan Purification

The cells undergo hydrolysis in alkali medium in the presence of salt inorder to cleave the proteoglycans and avoid ionic GAG/proteininteractions.

This treatment comprises the following steps:

1. Treatment with sodium hydroxide in saline medium: this step is aimedat destroying the cells and at cleaving the bonds between the heparinand its mother protein.

The step comprises the addition of 100 μl of 1 M NaOH and of 800 μl of0.5 M NaCl to a pellet of 10⁶ cells. The mixture thus obtained is heatedin a water bath at 80° C. for 30 minutes, and then sonicated for 5minutes before being neutralized with 1 N HCL.

2. Extraction: the hydrolyzed sample is loaded onto an anion exchangeresin column (SAX, Varian), which retains heparin. The column is washedthree times in Tris/HCl buffer, pH 7.4, containing 0.5 M NaCl in orderto eliminate the proteins and the other GAGs, in particular thedermatan. The heparin is then eluted with 1 ml of Tris/HCl buffer, pH7.4, containing 3 M NaCl.

3. Desalting/lyophilization: the elimination of the sodium chloride(necessary in order to be able to apply some of the analytical methodswhich are described below) is carried out by steric exclusionchromatography on SEPHADEX G10 gel, followed by conductimetry. Thecollected heparin fractions are then lyophilized so as to concentratethe sample.

Analysis by Polyacrylamide Gel Electrophoresis

This technique makes it possible to separate the GAGs according to theirsize and their charge, and constitutes a test for rapidly verifying thepresence or absence of heparin.

The purified preparation obtained as described above is loaded onto aTris/tricine polyacrylamide gel (gradient from 10 to 20%) for separatingmolecules of 30 to 1 kDa, in a proportion of 20 μl of preparation perdeposit. 25 ng of dermatan, and 25 ng of SPIM standard porcine heparin(4^(th) international standard for porcine heparin from intestinalmucosa), and of the heparin extracted from porcine mucosa and purifiedby treatment with sodium hydroxide and purification on anion exchangeresin under the same conditions as those described above are loaded ontothe same gel.

Double staining with a solution of alcian blue and then silver nitrateas described in AL-HAKIM and LINHARDT (Applied and TheoreticalElectrophoresis 1, 305-12, 1991) makes it possible to reveal theglycosaminoglycans (silver nitrate alone only reveals proteins).

The gels are then analyzed with a scanner (BIO-RAD) in order to quantifythe various GAGs. The heparin quantification limit is 10 ng per band.

The results of an experiment are summarized in Table 1 below, in whichthe amount of heparin produced by the cells is expressed as μg/10⁶cells. TABLE 1 Days of harvesting 3 4 5 6 7 10 11 14 Liver cells, dish2.6 3.5 4.4 6.5 3.7 4.2 — 8.1 Transfected liver 2.6 6.9 9.0 11.7 10.88.5 5.4 7.1 cells, dish Liver cells, flask 1.2 — — — — — — — Transfectedliver — 2.1 — — — — — — cells, flask

These results are also illustrated in FIG. 4 (curve=cell population;bars=heparin production).

FIG. 4 illustrates the heparin production during growth of the livermast cells in static culture in dishes.

The heparin concentrations generally observed are between 2 and 14 μgper 10⁶ cells, in static culture or in suspension.

EXAMPLE 2 Characterization of the Preparation of Heparin Obtained fromMast Cell Cultures

Disaccharide Profile by HPLC

The disaccharide composition makes it possible to differentiate theheparin from the other glycosaminoglycans.

The disaccharide profile of the glycosaminoglycans produced by the mastcells in culture was determined according to the method described byLINHARDT et al. (Biomethods, 9, 183-97, 1997).

The GAG preparation obtained as described in Example 1 above wasdepolymerized with a mixture of Flavo-bacterium heparinium heparinases(heparinases I, II and III, GRAMPIAN ENZYMES). The conditions used aredescribed in the publication by LINHARDT et al., mentioned above.

As a control, the SPIM standard heparin was depolymerized under the sameconditions.

Under these conditions, the depolymerization is complete and producesdisaccharides.

The main disaccharides, eight in number, which are either N-sulfated orN-acetylated, are represented in FIG. 5.

UV Detection

These disaccharides are separated and identified by HPLC, on an anionexchange column as described by LINHARDT et al. (mentioned above).

The results are illustrated in FIG. 6, representing the disaccharideprofile of the preparation of heparin produced by a flask culture offetal liver-derived mast cells (▪), compared to the disaccharide profileof the standard heparin (□).

These results show that all the disaccharides present in the SPIMreference porcine heparin are also present in the mast cell heparin,although in different proportions. The IS/IIS ratio is 3.7.

Fluorescence Detection

A similar method with fluorimetric detection makes it possible toquantify only the IS and IIS disaccharides, characteristic of heparin,and to calculate the ratio thereof.

The enzymatic depolymerization and the HPLC separation are carried outin the same way as that described above.

The separation is followed by a post-column derivatization, so as toform a fluorescent complex with guanidine.

The IS trisulfated disaccharide, which has the strongest response factorby this technique, is detected and quantified with respect to a solutionof standard heparin of known concentration.

The detection limit of the method is of the order of 5 ng/ml of heparinin the cell culture samples.

Table 2 below illustrates the IS/IIS ratio of cell cultures over time.TABLE 2 Days of harvesting 3 4 5 6 7 10 11 14 Liver cells, dish 1.9 1.61.6 1.4 1.4 1.3 — 1.4 Transfected liver 4.1 5 4.6 6.6 3.7 4.9 5.6 5.7cells, dish Liver cells, flask 2.3 — — — — — — — Transfected liver — 2.9— — — — — — cells, flask

EXAMPLE 3 Biological Characterization of the Heparin by Determination ofthe Anti-Xa and Anti-IIa Activities

Biological Activities

Inactivation of factors Xa and IIa is characteristic of heparin, andmakes it possible to differentiate it from heparan sulfate and fromdermatan.

The method used is that described in the European Pharmacopoeia, 3^(rd)edition (1997), monograph on low molecular weight heparins.

The reaction occurs in three steps:

-   1. ATIII+heparin→[ATIII−heparin]-   2.    [ATIII−heparin]+factor(excess)→([ATIII−heparin−factor]+factor(residual)-   3. factor(residual)+chromophore substrate→pNA

The amount of para-nitroaniline (pNA) released is measured at 405 nm. Itis inversely proportional to the amount of heparin.

The anti-Xa or anti-IIa activity is evaluated with respect to acalibration straight line established with the SPIM standard.

The sensitivity of the method is 0.006 IU/ml.

The results obtained are given in Table 3 below. TABLE 3 Days ofharvesting 3 4 5 6 7 10 11 14 Liver cells, dish: Anti-Xa 2.1 1.8 5.7 4.02.4 2.2 — 0.0 Anti-IIa — — — — — — — — Anti-Xa/anti- — — — — — — — — IIaratio Transfected liver cells, dish: Anti-Xa 44 11.5 11.7 12.3 11.4 13.011.6 12.9 Anti-IIa — — — — — — — — Anti-Xa/anti- — — — — — — — — IIaratio Liver cells, flask: Anti-Xa 0.7 — — — — — — — Anti-IIa 1.4 — — — —— — — Anti-Xa/anti- 0.6 — — — — — — — IIa ratio Transfected liver cells,flask: Anti-Xa — 3.1 — — — — — — Anti-IIa — 14 — — — — — — Anti-Xa/anti-— 0.2 — — — — — — IIa ratio

The anti-Xa or anti-IIa activity of the heparin obtained from mast cellsin culture was compared with the anti-Xa or anti-IIa activity,respectively, of the heparin obtained from porcine mucosa or of thestandard heparin. The results are illustrated in Table 4 below. TABLE 4Anti-Xa Anti-IIa Xa/IIa (IU/mg) (IU/mg) (IU/mg) Mast cell heparin 18 to3.1 14 to 3 0.2 to 1 Mucosal heparin 80 81 1 Standard heparin 180 180 1Characterization of the ATIII Binding

The binding between heparin and ATIII is demonstrated by a migrationshift using electrophoresis techniques as described in LEE and LANDER(Proc. Natl. Acad. Sci., 88, 2768-72, 1991).

The electrophoresis is carried out on a 0.8% agarose gel in a solutionof pH 3 (acetic acid/lithium hydroxide).

100 μl of ATIII (human origin; BIOGENIC) solution at decreasingconcentrations of 584 to 183 μg/ml are added to 100 μl of test sample.

100-μl deposits of sample are loaded. The migration is for 30 minutes at100 volts.

The gels are fixed with a solution of 0.1% hexadecyltrimethylammoniumbromide (CETAVLON-SIGMA).

Revelation is carried out with Azure A (0.08% in water).

The gels are scanned and interpreted with the QUANTITY ONE software(BIO-RAD).

The results are expressed as % heparin bound to ATIII.

The results obtained in the case of a flask culture of transfected livercells are illustrated in FIG. 7.

31% ATIII binding (theoretical value 33%) is observed in the presence ofstandard heparin (SPIM), and 27% ATIII binding in the presence of theheparin obtained from mast cells in culture (compound).

EXAMPLE 4 Culturing of Mast Cells in a Repeated Batch Bioreactor

An untransfected line of mast cells derived from porcine fetal liver wasused. The cells are seeded at a rate of 2.0 to 4.0×10 ⁵ cells per ml incomplete DMEM/F12 medium supplemented with porcine IL3 (2 ng/ml) andporcine SCF (80 ng/ml).

The bioreactor used has a volume of 2 liters of culture medium, theoxygen tension of the culture is maintained at between 20% and 40% ofsaturation, the pH is maintained between 7.0 and 7.4, and thetemperature is maintained at 37° C.+/−0.5° C. by circulation ofthermostated water in the bioreactor jacket. The culture is stirredusing a marine propeller, with a rate of between 80 and 150 rpm.

After culturing for 4 days, the cell density is 1.3×10⁶ cells/ml,corresponding to a doubling time of between 24 and 48 h. On the day ofharvesting, 80% of the culture is removed for the heparin extraction,and the remainder of the culture is kept in the bioreactor and dilutedwith fresh medium to a concentration of between 2.0 and 3.0×10⁵ cells/mlas described for a repeated-batch production operation. Three days afterdilution in repeated-batch mode, the cell density obtained is 9.0×10⁵cells/ml, corresponding to a doubling time of between 24 and 48 hoursand comparable to the first culturing (FIG. 8).

The heparin is purified as described in Example 1.

Purified heparin is then analyzed by HPLC, as described in Example 2,using the SPIM standard heparin as control.

Table 5 and FIG. 9 represent the disaccharide profile and the proportionof the serglycine (Gly-Ser) protein core of the preparation of heparinproduced by suspension-culturing of mast cells derived from porcinefetal liver (▪), compared to the profile obtained for the SPIM standardheparin (□).

Table 6 represents the N-acetylation, N-sulfation and O-sulfationprofile of the disaccharides of the heparin produced bysuspension-culturing of mast cells derived from porcine fetal liver,compared to that of the disaccharides of the standard SPIM heparin.TABLE 5 % Standard % Culture Gly-Ser 3.5 3.2 IVa 4 5.4 IVs 3.1 7.6 IIa3.1 4.4 IIIa 1.5 0.7 IIs 8.4 11.9 IIIs 7.2 17.1 Ia 1.3 0.2 Is 62 48.8

TABLE 6 Disaccharides % Standard % Culture Acetylated 11.8 10.72-O-sulfated 23 8 6-O-sulfated 42 42 N-sulfated 83 85 2-O-sulfated 84 776-O-sulfated 89 71 Sulfates/carboxylates 2.4 2.1

Similar results are obtained when a line of mast cells transfected withthe SV40 virus T antigen is used.

EXAMPLE 5 Production of Heparin in the Culture Supernatant Using aDegranulating Agent

The experiments were carried out on a line of untransfected fetal livermast cells.

On the 762^(nd) day (counting from the first culturing) the mast cellconcentration was adjusted to 2×10⁶ cells/ml, and the culture wasincubated for one hour in MEM medium comprising 4 μg/ml of the ionophoreA23187, which induces mast cell degranulation.

The total GAGs and the secreted GAGs produced by the cells arequantified by PAGE. FIG. 10 shows that 70 to 75% of the GAGs are foundin the supernatant after treatment with the ionophore A23187, versusapproximately 10% in the nontreated cells (0 μg/ml of A23187).

The mast cells for which the GAG harvesting was carried out on the762^(nd) day of culturing were placed in culture again. No loss ofviability or of growth rate was observed.

21 days later, these mast cells were subjected to a furtherdegranulation, and the GAGs were assayed as described above. A mast cellculture of the same age, which had not undergone degranulation on the762^(nd) day was used as a control.

The results are given in FIG. 10, which shows that the percentage ofGAGs secreted is comparable with that obtained during the firstdegranulation and also comparable to that obtained with the controlcells of the same age.

Similar results are obtained when a line of mast cells transfected withthe SV40 virus T antigen is used.

1. A method for producing heparin, which comprises: culturing mast cellsof porcine origin and recovering the heparin from the cultures obtained.2. The method as claimed in claim 1, wherein said mast cell cultures aremast cells lines of porcine origin.
 3. The method as claimed in claim 1,wherein said mast cells are derived from pig fetal bone marrow or pigfetal liver.
 4. The method as claimed in claim 1, said mast cells areserous mast cells.
 5. The method as claimed in claim 1, wherein saidmast cells are derived from a mast cell line selected from the groupconsisting of the line deposited with the CNCM [National Collection ofCultures of Microorganisms] on Oct. 17, 2001, under the number I 2735;the line deposited with the CNCM on Oct. 17, 2001, under the number I2736; and the line deposited with the CNCM on Oct. 17, 2001, under thenumber I
 2734. 6. A preparation of heparin which is prepared by theprocess as claimed in claim 1.